Targeted oligonucleotide conjugates

ABSTRACT

The present invention provides improved ingress of therapeutic and other moieties into cellular targets. In accordance with preferred embodiments, complexes are provided which carry primary moieties, chiefly therapeutic moieties, to such target cells. Such complexes preferably feature cell surface receptor ligands to provide specificity. Such ligands are preferably bound to primary moieties through polyfunctional manifold compounds.

This application is a continuation of Ser. No. 09/934,424 filed Aug. 21,2001 U.S Pat. No. 6,525,031 which is a divisional of Ser. No. 09/097,753filed Jun. 16, 1998, U.S. Pat. No. 6,300,319.

FIELD OF THE INVENTION

The present invention relates to complex compounds and methods for usingsuch complex compounds. The compounds of the invention are preferablyused in methods for targeting cellular receptors that facilitateendocytic processes. The present invention takes advantage of thisreceptor targeting to enhance the intracellular uptake of biologicallyactive compounds for therapeutic purposes.

BACKGROUND OF THE INVENTION

The use of synthetic, short, single stranded oligonucleotide sequencesto inhibit gene expression has evolved to the clinical stage in humans.It has been demonstrated that incorporation of chemically modifiednucleoside monomers into oligonucleotides can produce antisensesequences which can form more stable duplexes and can have highselectivity towards RNA (Frier et al., Nucleic Acids Research, 1997, 25,4429-4443). Two modifications that have routinely given high bindingaffinity together with high nuclease resistance are phosphorothioatesand methylphosphonates.

There are a number of desirable properties such as specificity, affinityand nuclease resistance that oligonucleotides should possess in order toelicit good antisense activity. The ability to selectively target and betaken up by diseased cells is another important property that isdesirable in therapeutic oligonucleotides. Natural oligonucleotides arepolyanionic and are known to penetrate cells at very low concentrations.Neutral oligonucleotides, such as the methylphosphonates, are taken upby cells at much higher concentrations. Although the processes by whichantisense oligonucleotides enter the cell membrane are not wellunderstood, there is substantial evidence for distinct mechanisms ofcell entry based on the electronic character of the antisense sequence.

Delivery of an antisense oligonucleotide to a specific, diseased cell isa very important area of active research. The majority of projectedantisense therapies are for viral infections, inflammatory and geneticdisorders, cardiovascular and autoimmune diseases and significantly,cancer. For example, in conventional chemotherapy, neoplasticity andvirus-related infections are treated with high drug concentrations,leading to overall high systemic toxicity. This method of treatment doesnot distinguish between diseased cells and healthy ones.

In the treatment of cancers, the ability of antisense agents todown-regulate or inhibit the expression of oncogenes involved intumor-transforming cells has been well documented in culture and animalmodels. For example, antisense inhibition of various expressed oncogeneshas been demonstrated in mononuclear cells (Tortora et al., Proc. Natl.Acad. Sci., 1990, 87, 705), in T-cells, in endothelial cells (Miller etal., P.O.P., Biochimie, 1985, 67, 769), in monocytes (Birchenall-Robertset al., Suppl. 1989, 13 (P.t. C), 18), in reticulocytes (Jaskulski, etal., Science 1988, 240, 1544)and in many other cell types, as generallyset forth in Table 1.

TABLE 1 INHIBITION OF MAMMALIAN GENE EXPRESSION INHIBITION OF EXPRESSIONCELL TYPE T cell receptor T cells Colony-stimulating factors Endothelialcells β-Globin Reticulocytes Multiple drug resistance MCF-1 cells cAMPkinase HL-60 cells bcl-2 L697 cells c-myb Mononuclear cells c-mycT-lymphocytes Interleukins Monocytes

Virally infected cell cultures and studies in animal models havedemonstrated the great promise of antisense and other oligonucleotidetherapeutic agents. Exemplary targets from such therapy includeeukaryotic cells infected by human immunodeficiency viruses(Matsukura etal., Proc. Natl. Acad. Sci., 1987, 84, 7706; Agrawal et al., Proc. Natl.Acad. Sci., 1988, 85, 7079), by herpes simplex viruses (Smith et al.,P.O.P., Proc. Natl. Acad. Sci., 1986, 83, 2787), by influenza viruses(Zerial et al., Nucleic Acids Res., 1987, 15, 9909) and by the humancytomegalovirus (Azad et al., Antimicrob. Agents Chemother., 1993, 37,1945). Many other therapeutic targets also are amenable to suchtherapeutic protocols.

The use of non-targeted drugs, to treat disease routinely causesundesirable interactions with non-diseased cells (Sidi et al., Br. J.Haematol., 1985, 61, 125; Scharenberg et al., J. Immunol., 1988, 28, 87;Vickers et al., Nucleic Acids Res., 1991, 19, 3359; Ecker et al.,Nucleic Acids Res., 1993, 21, 1853). One example of this effect is seenwith the administration of antisense oligonucleotide in hematopoieticcell cultures that exhibit non-specific toxicity due to degradativeby-products.

Other research efforts suggest that antisense oligonucleotides possessmore side effects in both in vitro and in vivo animal models. Forexample, non-complementary DNA sequences have been shown to interferewith cell proliferation and viral replication events through unknownmechanisms of action (Kitajima ibid). This reinforces the desirabilityof oligonucleotides that are specifically targeted to diseased cells.

When phosphodiester oligonucleotides are administered to cell cultures,a concentration of typically about 1 mmol is required to see antisenseeffects. This is expected since local endonucleases and exonucleasescleave these strands effectively and only 1-2% of the totaloligonucleotide concentration becomes cell-associated (Wickstorm et al.,Proc. Natl. Acad. Sci. 1988, 85, 1028; Wu-Pong et al., Pharm. Res. 1992,9, 1010). If chemically modified oligonucleotides, such as thephosphorothioates or methylphosphonates are used, the observed antisenseeffects are anywhere between 1 and 100 μM. This observed activity isprimarily due to the relatively slow cellular uptake ofoligonucleotides. There is evidence which suggests that a 80 kiloDalton(kDa) membrane receptor mediates the endocytic uptake of natural andphosphorothioate oligonucleotides in certain type of cells. Other dataquestion the existence of such a link between receptor-mediatedoligonucleotide uptake and internalization of oligonucleotides. Forexample, inhibitors of receptor-mediated endocytosis have no effect onthe amount of oligonucleotide internalized in Rauscher cells (Wu-Pong etal., Pharm. Res., 1992, 9, 1010). For uncharged methylphosphonates, itwas previously believed that internalization of such agents occurred bypassive diffusion (Miller et al., Biochemistry 1981, 20, 1874). Thesefindings were disproved by studies showing that methylphosphonates takeup to 4 days to cross phospholipid bilayers, which correlates well withthe fate of internalization of natural oligonucleotides (Akhtar et al.,Nucleic Acids Res., 1991, 19, 5551).

Increased cellular uptake of antisense oligonucleotides by adsorptiveendocytosis can be obtained by liposome encapsulation. In one study,researchers showed that a 21-mer complementary to the 3′-tat spliceacceptor of the HIV-1 was able to markedly decrease the expression of ap24 protein while encapsulated into a liposome containingdiastearoylphosphatidylethanol-amine (Sullivan et al., Antisense Res.Devel., 1992, 2, 187). Many other examples have been reported, includingpH-sensitive liposomes (Huang et al., Methods Enzymol., 1987, 149, 88)which are well detailed in several good review articles (Felgner et al.,Adv. Drug Deliv. Rev., 1990, 5, 163 and Farhood et al., N.Y. Acad. Sci.,1994, 716, 23). When phosphorothioate oligonucleotides, that arecomplementary to the methionine initiation codon of human intracellularadhesion molecule-1, were encapsulated, a 1000-fold increase ofantisense potency was seen relative to the non-encapsulatedphosphorothioate oligonucleotide (Bennett et al., Mol. Pharmacol., 1992,41, 1023). The oligonucleotide delivery systems are good for in vitrocell systems, but have not been shown to be widely applicable to in vivostudies, due to rapid liposome destabilization and non-specific uptakeby liver and spleen cells.

Other, non-specific oligonucleotide uptake enhancements attend attachinghydrophobic cholesterol (Letsinger et al., Proc. Natl. Acad. Sci., 1989,86, 6553) type or phospholipid type molecules (Shea et al., NucleicAcids Res., 1990, 18, 3777) to the oligonucleotides. It has been shownthat the coupling of a single cholesterol moiety to an antisenseoligonucleotide increases cellular uptake by 15-fold (Boutorin et al.,FEBS Lett., 1989, 254, 129). When the cationic polymeric drug carrierpoly(L-lysine) is conjugated to oligonucleotide sequences, a markedincrease of non-specific oligonucleotide cellular uptake occurs(Lemaitre et al., Proc. Natl. Acad. Sci., 1987, 84, 648; Leonetti etal., Gene 1988, 72, 323; Stevenson et al., J. Gen. Virol., 1989, 70,2673). This cationic polymer has been used to deliver several types ofdrugs with cellular uptake mediated by an endocytic-type mechanism.However, the high molecular weight polylysine is cytotoxic even at lowconcentrations.

Cell surface receptors are good candidates to serve as selective drugtargets. The presence of specific receptors implies that naturalendogenous ligands are also present. It is the complexation of theligand with the appropriate receptor that elicits a cascade of cellularevents leading to a desired function. An oligonucleotide drug linked tosuch an endogenous ligand or a synthetic ligand of equal affinitytowards the receptor in question, is considered “targeted” to thereceptor.

The potential of carbohydrate drug targeting has become increasinglyapparent (Shen et al., N.Y. Acad. Sci., 1987, 507, 272; Monsigny et al.,N.Y. Acad. Sci., 1988, 551, 399; Karlsson et al., TIPS 1991, 12, 265) asan alternate method for site-specific drug delivery. Complexcarbohydrates are involved in many cellular recognition processes suchas adhesion between cells, adhesion of cells to the extracellularmatrix, and specific recognition of cells (Ovarian egg with sperm) byone another (Yamada, K. M., Annu., Rev. Biochem., 1983, 52, 761;Edelman, G. M., Annu. Rev. Biochem., 1985, 54, 135; Hook et al., Annu.Rev. Biochem., 1984, 53, 847; Florman, H. M., Cell, 1985, 41, 313. It isalso known that the concentrations of various glycosylated proteins thatcirculate in the blood are constantly regulated by cells in varioustissues. Nature controls and regulates such diverse functions with theaid of specific proteins appearing at the surface of various cells,which have the ability to decode the information found in complexcarbohydrate structures. These proteins are collectively called lectinsand act as receptors for carbohydrates (Goldstein et al., Nature, 1980,285, 66). Many endogenous lectins are expressed at the surface of normaland malignant cells and are involved in many poorly understoodbiological processes.

The structural information obtained from a large number of mammalianlectins has led to their classification into several families.

TABLE 2 PROPERTIES OF C-TYPE AND S-TYPE ANIMAL LECTINS PROPERTY C-TYPELECTINS S-TYPE LECTINS Ca⁺⁺⁻dependance Yes No Solubility Variable Buffersoluble Location Extracellular Intracellular/extra State of cysteinesDisulfides cellular Carbohydrate Different types Free thiols specificityMostly β- galactosides

The C-lectins or calcium-dependent lectins possess carbohydraterecognition domains (CRDs) of the 115-134 amino acids which contain 18highly conserved and 14 invariant residues (Drickamer, K., J. Biol.Chem., 1988, 263, 9557; Drickamer, K., Curr. Opin. Struc. Biol., 1993,3, 393; Drickamer, K., Biochemical Society Transactions, 1993, 21, 456).The C-lectins are interesting from a pharmacological point of view sincethey recognize specific carbohydrates and immediately endocytose thereceptor-bound glycoprotein complex via coated pits and vesicles.

These vesicles which are also referred to as endosomes, bring thereceptor-glycoprotein complex to other cellular compartments, called thelysozomes, where protein degradation occurs (Breitfeld et al., Int. Rev.Cytol., 1985, 97, 4795). The range of C-type lectin carbohydratespecificity differs form cell to cell and from tissue to tissue.

The first membrane lectin was characterized on hepatocyte liver cells(Van Den Hamer et al., J. Mol. Biol., 1970, 245, 4397). The hepaticasialoglycoprotein receptor (ASGP-R) was isolated by Ashwell and Harford(Ashwell, G.; Herford, J., Ann. Rev. Biochem. 1982, 51, 531; Schwartz,A. L., CRC Crit. Rev. Biochem., 1984, 16, 207). These lectinsinternalized efficiently and cleared plasma levels from ceruloplasminwhich contained abnormally truncated N-oligosaccharides lacking theterminal sialic acid residues. Other artificial molecules which haveterminal galactose or N-acetylgalactosamine residues have been found tobind with high affinity to this lectin. This unique specificity betweenthe exposed galactose units and the ASGP-R suggested the design andtesting of glycotargeting systems and the use of lectins as specificdrug delivery targets.

TABLE 3 MEMBRANE SPANNING C-TYPE LECTINS NAME TISSUE SUGAR SPECIFICITYASGP-R (type II) Liver Hepatocytes Galactose and N-acetylgalactoseamine¹Placental Placenta Fucose and mannose² (type receptor II) MacrophageLiver Kupffer Galactose and receptor cells type II) N-acetylgalactoseamine Kupffer cell Liver Kupffer Galactose and fucose receptorcells (type II) IgE Fc receptor B cells Galactose⁵ (type II) P-selectinPlatelets Fucose and sialic acid (type IV) E-selectin Endothelial cellsFucose and sialic (type (type IV) IV) acid⁷ L-selectin Leukocytes Fucoseand sialic acid⁸ (type IV) Mannose receptor Macrophages Mannose andfucose⁹ (type VI) ¹Spiess et al., J. Biol. Chem., 1985, 260, 1979.²Curtis et al., Proc. Natl. Acad. Sci., 1992, 89, 8356. ³Ii, et al., J.Biol. Chem., 1990, 265, 11295. ⁴Hoyle et al., J. Biol. Chem., 1988, 263,7487. ⁵Kikutani et al., Cell, 1986, 47, 657. ⁶Johnston et al., Cell,1989, 56, 1033. ⁷Bevilacqua et al., Science, 1989, 243, 1160. ⁸Laskyk etal., Cell, 1989, 56, 1045. ⁹Taylor et al., J. Biol. Chem., 1992, 267,1719.

Many other cell lines, some summarized in Table 2, have surfacecarbohydrate-type receptors that mediate uptake of various ligands(Drickamer, K., Cell 1991, 67, 1029). Immune cells like monocytes andmacrophages possess a number of surface glycoproteins that enable themto interact with invading micro-organisms (Gordon et al., J. Cell Sci.Suppl., 1988, 9, 1). Drugs need to be carried to target cells via acarrier or high affinity ligand which is attached to the drug. Thedifferent carriers for glycotargeting can be glycoproteins orneoglycoproteins, (glycopeptides or neoglycopeptides) and asglycosylated polymers.

The in vitro glycotargeting principle is relatively simple, but its invivo applicability is difficult. Synthetic efforts have generatedliposomes (also referred as immunoliposomes) and polylysine carriers, inwhich antibodies and some carbohydrate conjugate ligands have beencovalently attached on the outer bilayer. For example, when naturaloligonucleotides complementary to the translation initiation region ofVSV N protein mRNA were encapsulated with liposomes whose outer membranehad several H2K-specific antibodies to L929 cells, there was a markeddecrease in viral replication only within L929 infected cells (Leonettiet al., Proc. Natl. Acad. Sci., 1990, 87, 2448). Receptor-mediatedendocytic mechanisms have been exploited by attachment of cell-specificligands and antibodies to polylysine polymers. For example, c-mybantisense oligonucleotides conjugated with polylysine-folic acid (Citroet al., Br. J. Cancer 1992, 69, 463) or polylysine-transferrin (Citro etal., Proc. Natl. Acad. Sci., 1992, 89, 7031) targets were found tobetter inhibit HL-60 leukemia cell line proliferation thanoligonucleotides without conjugated carriers. Another promisingpolylysine-asialoorosomucoid carrier was conjugated withphosphorothioate oligonucleotides complementary to the polyadenylationsignal of Hepatitis B virus (Wu, G. Y.; Wu, C. H., J. Biol. Chem., 1992,267, 12436).

Methods have been previously developed that utilize conjugates toenhance transmembrane transport of exogenous molecules. Ligands thathave been used include biotin, biotin analogs, other biotinreceptor-binding ligands, folic acid, folic acid analogs, and otherfolate receptor-binding ligands. These materials and methods aredisclosed in U.S. Pat. No. 5,108,921, issued Apr. 28, 1992, entitled“Method for Enhanced Transmembrane Transport of Exogenous Molecules”,and U.S. Pat. No. 5,416,016, issued May 16, 1995, entitled “Method forEnhancing Transmembrane Transport of Exogenous Molecules”, thedisclosures of which are herein incorporated by reference.

These immunoliposomes and antibody-polymer targeting exhibited no invivo activity. With similar drawbacks as their non-specificcounterparts, the immunoliposome-drug complexes are mostly immunogenicand are phagocytosed and eventually destroyed in the lysosomecompartments of liver and spleen cells. As for theantibody-polylysine-drug complexes, they have shown substantial in vitrocytotoxic activity (Morgan et al., J. Cell. Sci., 1988, 91, 231). Othercarriers are glycoproteins. On such large structures, a few drugmolecules can be attached. The glycoprotein-drug complexes cansubsequently be desilylated, either chemically or enzymatically, toexpose terminal galactose residues.

Glycoproteins and neoglycoproteins are recognized by lectins such as theASGP-R. Glycoproteins having a high degree of glycosylationheterogeneity are recognized by many other lectins making targetspecificity difficult (Spellman, N. W., Anal. Chem., 1990, 62, 1714).Neoglycoproteins having a high degree of homogeneity exhibit a higherdegree of specificity for lectins especially the ASGP-R. Manyexperimental procedures which are used to couple sugars to proteins havebeen reviewed by Michael Brinkley (Brinkley, M., Bioconjugate Chem.,1992, 3, 2). These neoglycoproteins may mimic the geometric organizationof the carbohydrate groups as in the native glycoprotein and should havepredictable lectin affinities. Successful in vitro delivery ofAZT-monophosphate, covalently attached to a human serum albumincontaining several mannose residues was achieved in human T4 lymphocytes(Molema et al., Biochem. Pharmacol., 1990, 40, 2603).

Examples of antisense oligonucleotide-neoglycoprotein complexes havebeen previously reported (Bonfils et al., Nucleic Acids Res., 1992, 20,4621). The authors mannosylated bovine serum albumin and attached,covalently from the 3′-end, a natural oligonucleotide sequence. Theoligonucleotide-neoglycoprotein conjugate was internalized by mousemacrophages in 20-fold excess over the free oligonucleotide.Biotinylated oligonucleotides, were also disclosed which werenon-covalently associated with mannosylated streptavidin (Bonfils etal., Bioconjugate Chem., 1992, 3, 277). Such complexes were also betterinternalized by macrophages. Other successful examples consisted ofantisense oligonucleotides which were non-covalently associated withasialoglycoprotein-polylysine conjugates. Such oligonucleotideconjugates were found to internalize more efficiently into hepatocytes(Bunnel et al., Somatic Cell Molecular Genetics, 1992, 18, 559; Reiniset al., J. Virol. Meth., 1993, 42, 99) and into hepatitis B infectedHepG2 cells (Wu, G. Y.; Wu, C. H., J. Biol. Chem., 1992, 267, 12436).

Polymeric materials have been assessed as drug carriers and three ofthem, dextrans, polyethyleneglycol (PEG) andN-(2-hydroxypropyl)methacrylamide (HMPA) co-polymers, have beensuccessfully applied in vivo (Duncan, R., Anticancer Drugs, 1992, 3,175). This research has been focused mainly at treatments for cancer andas a requisite the size of the compounds are between 30-50 kDa to avoidrenal excretion (Seymour, L. W., Crit. Rev. Ther. Drug Carrier Syst.,1992, 9, 135).

In order to examine the chemistry and related methodologies involvingthe preparation of glycoprotein-drug and neoglycoprotein-drugglycoconjugates, certain groups have investigated the use of simplerhigh affinity ligands for specific drug delivery. Initially, severalsugars were attached to small peptides in an attempt to obtain mimics ofmultivalent N-linked oligosaccharides (Lee, R. T., Lee, Y. C.,Glycoconjugate J., 1987, 4, 317; Plank et al., Biconjugate Chem., 1992,3, 533; Haensler et al., Bioconjugate Chem., 1993, 4, 85).

Other groups investigated the use of sugar clusters lacking a proteinbackbone and eventually used low molecular weight N-linkedoligosaccharides with a minimum carbohydrate population to bind withhigh affinity to lectins as the ASGP-R. Branched N-linkedoligosaccharide-drug conjugates can be used instead ofneoglycoprotein-drug complexes. The total synthesis of branched N-linkedoligosaccharides is still a difficult task, however they could beobtained by enzymatic cleavage from protein backbones (Tamura et al.,Anal. Biochem., 1994, 216, 335). This method requires expensivepurifications and only generates low quantities of chemically definedcomplex oligosaccharides. The affinity of N-linked oligosaccharideclusters towards many lectins has been demonstrated and has helpedresearchers to locate different new mammalian lectins in animals (Chiuet al., J. Biol. Chem., 1994, 269, 16195).

One process of increasing the intracellular oligonucleotideconcentration is via receptor-mediated endocytic mechanisms. This noveldrug targeting concept has been demonstrated in vitro by several groups.Oligonucleotides have been attached to glycoproteins, neoglycoproteinsand neoglycopolymers possessing a defined carbohydrate population which,in turn, are specifically recognized and internalized by membranelectins. To the best of our knowledge in vivo applicability ofoligonucleotide-carbohydrate conjugates has not been previouslydemonstrated.

It has also been shown in in vitro experiments that syntheticneoglycoproteins containing galactopyranosyl residues at non-reducingterminal positions are recognized by the ASGP-R with increasing affinityas the number of sugar residues per molecule is increased (Kawaguchi etal., J. Biol. Chem. 1981, 256, 2230).

OBJECTS OF THE INVENTION

As in apparent, there exists a need for an improved method of selectivedelivery of biologically active compounds such as antisenseoligonucleotides to specific cells. This invention is directed toproviding methods to effect such delivery.

SUMMARY OF THE INVENTION

The present invention provides complexes and methods for using suchcomplexes. The complex forms are useful for enhancing the intracellularuptake of biologically active compounds (primary compounds). The complexcompounds of the invention are prepared having the component parts shownbelow:

wherein the primary moiety is a nucleotide, nucleoside, oligonucleotideor oligonucleoside; each of said linkers are, independently, bi- ortrifunctional; said manifold is derivatized at a plurality of sites;eachof said cell surface receptor ligands is a carbohydrate; and n is aninteger from 2 to about 8.

Preferably, at least two cell surface receptors are individually linkedby linker groups to a manifold compound which is further linked to aprimary compound. The cell surface receptor ligands impart affinity tothe complexes for cells having surface receptors that recognize theselected cell surface receptor ligands. This interaction is believed totrigger endocytosis of the complex, resulting in an increased uptake bythe cell of the primary compound.

In one embodiment of the invention primary compounds are selected to beoligonucleotides or oligonucleosides. Attachment of oligonucleotides oroligonucleosides to a manifold compound, can be conveniently made at the5′ or 3′ phosphate of the 5′ or 3′ terminal nucleotide or nucleoside ofthe oligonucleotide or oligonucleoside. Alternatively, the phosphategroup can be introduced as part of the linker group attached to themanifold moiety. Such a coupling is made by selecting the terminus ofthe linker to be a hydroxyl group and converting it to aphosphoramidite. The phosphoramidite can then be reacted with anunblocked 2′, 3′ or 5′ hydroxyl group of an oligonucleotide oroligonucleoside.

Manifold species as used in the present invention can include a widevariety of compounds that have functional groups or sites that can belinked by linker groups to a primary compound together with a cellsurface receptor ligand, or, preferably ligands. In one embodiment apolycyclic molecule may be selected as the manifold compound, as can beillustrated for cholic acid. In other embodiments a smaller, monocyclicmanifold compound can be selected, such as phenyl or cyclohexyl.

Manifold compounds can also comprise branched chain aliphatic compoundsthat have the funtionalities available for linking. Also, combinatorialchemistry techniques are known to utilize numerous compounds that can beused as manifold compounds. Many combinatorial scaffolds are ammenablefor use as manifold compounds by virtue of their multiple reactivesites, which can be subjected to various orthogonal protection schemes.Preferable reactive sites include hydroxyl groups, carboxylic acidgroups, amino groups and thiol groups.

Linker groups that are preferred for use in the present invention can beselected for a variety of chemical reasons. If the primary compound isan oligonucleotide or oligonucleoside, a linker can be convenientlychosen having a secondary hydroxyl group and/or a primary hydroxyl groupwith an additional functionality such as an amino, hydroxyl, carboxylicacid, or thiol group. The additional functionality can be used to attachone end of the linker group to the manifold moiety by for example anamide linkage. The secondary and or primary hydroxyl groups can be usedto prepare a DMT or DMT phosphoramidite as illustrated in the Examplesection. This enables the attachment to an oligonucleotide oroligonucleoside to a solid support or to the 2′, 3′ or 5′ position or aribosyl group. This will allow variability of the placement of theconjugated manifold compound to the primary compound. In a preferredembodiment an oligonucleotide is prepared using standard automated solidsupport protocols as is well known in the art and the conjugatedmanifold compound is coupled as the last step to the 5′-O position ofthe completed oligonucleotide or oligonucleoside. Cleavage from thesolid support will give the complex compound.

For use in antisense and similar methodologies, oligonucleotides andoligonucleosides of the invention preferably comprise from about 10 toabout 30 subunits. It is more preferred that such oligonucleosidescomprise from about 15 to about 25 subunits. As will be appreciated, asubunit is a base and sugar combination suitably bound to adjacentsubunits through, for example, a phosphorous-containing (e.g.,phosphodiester) linkage or some other linking moiety. The nucleosidesneed not be linked in any particular manner, so long as they arecovalently bound. Exemplary linkages are those between the 3′- and5′-positions or 2′- and 5′-positions of adjacent nucleosides. Exemplarylinking moieties are disclosed in the following references: Beaucage, etal., Tetrahedron 1992, 48, 2223 and references cited therein; and U.S.patent applications: Ser. No. 703,619, filed May 21, 1991; Ser. No.903,160, filed Jun. 24, 1992; Ser. No. 039,979, filed Mar. 20, 1993;Ser. No. 039,846, filed Mar. 30, 1993; and Ser. No. 040,933, filed Mar.31, 1993. Each of the foregoing patent applications are assigned to theassignee of this invention. The disclosure of each is incorporatedherein by reference.

In other embodiments of the invention, primary compounds compriseoligonucleotides or oligonucleosides attached through a linking moietyto the manifold moiety such as by a free 2′-, 3′-, or 5′-hydroxyl group.Such attachments are prepared by, for example, reacting nucleosidesbearing at least one free 2′-, 3′-, or 5′-hydroxyl group under basicconditions with a linking moiety having a leaving group such as aterminal L—(CH₂)— etc. function, where L is a leaving group.Displacement of the leaving group through nucleophilic attack of (here)an oxygen anion produces the desired derivative. Leaving groupsaccording to the invention include but are not limited to halogen,alkylsulfonyl, substituted alkylsulfonyl, arylsulfonyl, substitutedarylsulfonyl, hetercyclcosulfonyl or trichloroacetimidate. A morepreferred group includes chloro, fluoro, bromo, iodo,p-(2,4-dinitroanilino)benzenesulfonyl, benzenesulfonyl, methylsulfonyl(mesylate), p-methylbenzenesulfonyl (tosylate), p-bromobenzenesulfonyl,trifluoromethylsulfonyl (triflate), trichloroacetimidate, acyloxy,2,2,2-trifluoroethanesulfonyl, imidazolesulfonyl, and2,4,6-trichlorophenyl, with bromo being preferred.

Suitably protected nucleosides can be assembled into an oligonucleosidesaccording to many known techniques. See, e.g., Beaucage, et al.,Tetrahedron 1992, 48, 2223.

A wide variety of linker groups are known in the art that will be usefulin the attachment of primary compounds to manifold compounds. Many ofthese are also useful for the attachment of cell surface receptorligands to the manifold compound. A review of many of the useful linkergroups can be found in Antisense Research and Applications, S. T. Crookeand B. Lebleu, Eds., CRC Press, Boca Raton, Fla., 1993, p. 303-350. Adisulfide linkage has been used to link the 3′ terminus of anoligonucleotide to a peptide (Corey, et al., Science 1987, 238, 1401;Zuckermann, et al., J. Am. Chem. Soc. 1988, 110, 1614; and Corey, etal., J. Am. Chem. Soc. 1989, 111, 8524). Nelson, et al., Nuc. Acids Res.1989, 17, 7187 describe a linking reagent for attaching biotin to the3′-terminus of an oligonucleotide. This reagent,N-Fmoc-O-DMT-3-amino-1,2-propanediol is now commercially available fromClontech Laboratories (Palo Alto, Calif.) under the name 3′-Amine on. Itis also commercially available under the name 3′-Amino-Modifier reagentfrom Glen Research Corporation (Sterling, Va.). This reagent was alsoutilized to link a peptide to an oligonucleotide as reported by Judy, etal., Tetrahedron Letters 1991, 32, 879. A similar commercial reagent(actually a series of such linkers having various lengths ofpolymethylene connectors) for linking to the 5′-terminus of anoligonucleotide is 5′-Amino-Modifier C6. These reagents are availablefrom Glen Research Corporation (Sterling, Va.). These compounds orsimilar ones were utilized by Krieg, et al., Antisense Research andDevelopment 1991, 1, 161 to link fluorescein to the 5′-terminus of anoligonucleotide. Other compounds such as acridine have been attached tothe 3′-terminal phosphate group of an oligonucleotide via apolymethylene linkage (Asseline, et al., Proc. Natl. Acad. Sci. USA1984, 81, 3297).

Oligonucleotides have been prepared on solid support and then linked toa peptide via the 3′ hydroxyl group of the 3′ terminal nucleotide(Haralambidis, et al., Tetrahedron Letters 1987, 28, 5199). An Aminolink2 (Applied Biosystems, Foster City, Calif.) has also been attached tothe 5′ terminal phosphate of an oligonucleotide (Chollet, Nucleosides &Nucleotides 1990, 9, 957). This group also used the bifunctional linkinggroup SMPB (Pierce Chemical Co., Rockford, Ill.) to link an interleukinprotein to an oligonucleotide.

In another embodiment of the invention, linker moieties are used toattach manifold groups to the 5 position of a pyrimidine (Dreyer, etal., Proc. Natl. Acad. Sci. USA 1985, 82, 968). Fluorescein has beenlinked to an oligonucleotide in the this manner (Haralambidis, et al.,Nucleic Acid Research 1987, 15, 4857) and biotin (PCT applicationPCT/US/02198). Fluorescein, biotin and pyrene were also linked in thesame manner as reported by Telser, et al., J. Am. Chem. Soc. 1989, 111,6966. A commercial reagent, Amino-Modifier-dT, from Glen ResearchCorporation (Sterling, Va.) can be utilized to introduce pyrimidinenucleotides bearing similar linking groups into oligonucleotides.

Cholic acid linked to EDTA for use in radioscintigraphic imaging studieswas reported by Betebenner, et.al., Bioconjugate Chem. 1991, 2, 117.

In a preferred embodiment of the present invention, novel complexcompounds are prepared having oligonucleotide conjugates that are usefulfor oligonucleotide antisense drug targeting of, for example, thecarbohydrate recognition domains (CRD) found on theasiologlycoprotein-receptor (ASGP-R). These complex compounds wereprepared according to the principles which govern the specificity of the{ligand-[ASGP-R]} complex. Simple carbohydrates and Glycoconjugateshaving only one linked saccharide moiety show a slight affinity for thereceptor (Lee et al., Biol. Chem., 1983, 258, 199). For instance,glycoconjugates having monovalent ligands such as galactose, lactose ormonoantennary galactosides (one carbohydrate group attached via alinkage to the scaffold) bind to this ASGP-R with a millimolardissociation constant. When binary oligosaccharides (two carbohydrategroups each attached via a linkage to the scaffold) are used, thedissociation constants are in the micromolar range. This translates to athree order of magnitude higher affinity. When trinary oligosaccharides(three carbohydrate groups each attached via a linkage to the scaffold)are tested, the dissociation constants are in the nanomolar range.

Based on dissociation constants, e.g. higher for 3 carbohydrate groups,trinary oligosaccharides were preferably synthesized, each having threecarbohydrate groups independently linked to a scaffold which was furtherlinked to an oligonucleotide. The resulting low molecular weightoligonucleotide conjugates were easily amenable to automated DNAsynthesis methodology. The oligonucleotide conjugates each consist of atleast four distinct moieties, scaffold, carbohydrate attaching linker,oligonucleotide attaching linker, and carbohydrate.

Initially, cholic acid was chosen as a scaffold. Cholic acid was chosenbecause it is a natural product in mammalian systems, does not form atoxic metabolite and because it is commercially available at low cost.Another reason for choosing cholic acid was that this steroidal scaffoldwould be a good anchor for linked carbohydrates, separating the pointsof attachment and reducing any steric interference between them.Increasing the distance between points of attachment of the linkedcarbohydrates would increase the degree of freedom and reduce the lengthrequirement of the linker to obtain high affinity with the receptor.

Galactose and lactose were initially chosen as carbohydrate moietiessince they are recognized by the carbohydrate recognition domains (CRD)found on the asiologlycoprotein-receptor (ASGP-R).

Aminocaproate, derived from commercially available N-Fmoc-ε-aminocaproicacid, was chosen as the carbohydrate linker and its length was based onpreviously reported experimental evidence (Biessen et al., J. Med.Chem.,1995, 38, 1538). The previously reported results indicated that clustergalactosides between 10 and 20 Å in length were high affinity substratesfor the hepatic ASGRP-R. The length of the linking group was initiallychosen to be eight atoms long because in conjunction with the largerarea scaffold being used the result may be better positioning of thecarbohydrate components towards the CRD's of ASGP-R.

After the cholic acid scaffold has been linked to each of thecarbohydrates and a linker group is deblocked and ready for attachmentan oligonucleotide is coupled. The preferred method of coupling of theconjugate to an oligonucleotide is to perform the coupling while thefull length oligonucleotide is bound to solid support. The conjugate iscoupled to the oligonucleotide followed by cleavage of the final productfrom the solid support. This cleavage step also removes acetylprotecting groups present on any hydroxyl groups that were previouslyprotected especially on any saccharide moieties.

The oligonucleotide analog is tested for affinity towards the ASGP-Rexpressed on several cells. The binding of the oligonucleotide conjugateto the ASGP-R should initiate the internalization process and increasethe intracellular concentration of the selected oligonucleotide.

It will be appreciated that modified nucleotide moieties may also beuseful in connection with embodiments of this invention. Thus, a widevariety of chemical modifications may be employed throughout the nucleicacids. Thus modifications of pyrimidine or purine bases, substitutionsat the 2′ position, alteration of inter-nucleoside linkages,carbohydrate ring substitutions and positional variations may all beemployed.

Teachings regarding the synthesis of modified oligonucleotides may befound in the following U.S. patents or pending patent applications, eachof which is commonly assigned with this application: U.S. Pat. Nos.5,138,045 and 5,218,105, drawn to polyamine conjugated oligonucleotides;U.S. Pat. No. 5,212,295, drawn to monomers for the preparation ofoligonucleotides having chiral phosphorus linkages; U.S. Pat. Nos.5,378,825 and 5,541,307, drawn to oligonucleotides having modifiedbackbones; U.S. Pat. No. 5,386,023, drawn to backbone modifiedoligonucleotides and the preparation thereof through reductive coupling;U.S. Pat. No. 5,457,191, drawn to modified nucleobases based on the3-deazapurine ring system and methods of synthesis thereof; U.S. Pat.No. 5,459,255, drawn to modified nucleobases based on N-2 substitutedpurines; U.S. Pat. No. 5,521,302, drawn to processes for preparingoligonucleotides having chiral phosphorus linkages; U.S. Pat. No.5,539,082, drawn to peptide nucleic acids; U.S. Pat. No. 5,554,746,drawn to oligonucleotides having β-lactam backbones; U.S. Pat. No.5,571,902, drawn to methods and materials for the synthesis ofoligonucleotides; U.S. Pat. No. 5,578,718, drawn to nucleosides havingalkylthio groups, wherein such groups may be used as linkers to othermoieties attached at any of a variety of positions of the nucleoside;U.S. Pat. Nos. 5,587,361 and 5,599,797, drawn to oligonucleotides havingphosphorothioate linkages of high chiral purity; U.S. Pat. No.5,506,351, drawn to processes for the preparation of 2′-O-alkylguanosine and related compounds, including 2,6-diaminopurine compounds;U.S. Pat. No. 5,587,469, drawn to oligonucleotides having N-2substituted purines; U.S. Pat. No. 5,587,470, drawn to oligonucleotideshaving 3-deazapurines; U.S. Pat. No. 5,223,168, issued Jun. 29, 1993,and U.S. Pat. No. 5,608,046, both drawn to conjugated 4′-desmethylnucleoside analogs; U.S. Pat. Nos. 5,602,240, and 5,610,289, drawn tobackbone modified oligonucleotide analogs; and U.S. patent applicationSer. No. 08/383,666, filed Feb. 3, 1995, and U.S. Pat. No. 5,459,255,drawn to, inter alia, methods of synthesizing2′-fluoro-oligonucleotides.

It is to be understood that all modifications to nucleotides,nucleosides at oligonomers thereof are encompassed within theirrespective definitions.

Antisense oligonucleotides are conversely synthesized using automatedDNA synthetic methodology. Therefore, small glycotargeting systems,which can be incorporated into the last cycle of an automated DNAsynthesis, are preferred.

EXAMPLE 1 Synthesis of 2,3,4,5-aceto-1-bromo-α-D-galactose

The title compound was synthesized using slightly modified, knownprocedures to generate the acetobromo-galactose (Methods in CarbohydrateChemistry, Wistler and Wollfrom, Eds, Academic Press; New York, 1962;Vol. 1-6). Dry D-galactose was reacted with acetic anhydride (20 eq.) inthe presence of a catalytic amount of DMAP (0.1 eq.) in dry pyridine at0° C. After 1 hour at 0° C., the reaction mixture was allowed to warm toroom temperature. After 6 hours had passed the thick solution was pouredinto a rapidly stirred solution of ice-water (500 mL). A stickyprecipitate developed and was extracted with ethyl acetate.1,2,3,4,5-pentaacetyl-α-D-galactose was obtained as a slightly yellowoil in 89% yield.

The 1,2,3,4,5-pentaacetyl-α-D-galactose was used without furtherpurification in the next step by treatment with HBr (5 molar eq., 30%solution in glacial acetic acid). After one hour, the HBr and the aceticacid was removed, giving rise to the title α-bromogalactoside as a thickbrown oil in 94% yield which was used for the next step without furtherpurification.

EXAMPLE 2 Synthesis of α-D-lactosyl Acid Bromide

Following the procedures illustrated for Example 1 above, dry D-lactosewas transformed to octaacetylated D-lactose, which was obtained as a20:80 mixture of α/β-anomers (α-anomer: doublet, 6.19 ppm,J_(H1′-H2′)=3.7 Hz, H1′; β-anomer: doublet, 5.1 ppm, J_(H1′-H2′)=8.4 Hz,H1′) in 76% yield. The octaacetate was used without further purificationby treatment with a solution of HBr (5 molar eq., 30% solution inglacial acetic acid). After the lactosyl compound dissolved (^(˜)4-5min.), the HBr and the acetic acid were quickly removed to avoidhydrolysis of the β(1-4)-O-glycosidic linkage. The title compound wasobtained as a thick brown oil in 97% yield. The title compound was usedwithout further purification in the next step.

EXAMPLE 3 Synthesis of peracetylated-β-azidogalactose

2,3,4,5-Aceto-1-bromo-α-D-galactose was reacted under phase transfercatalysis (PTC) conditions previously reported (Tropper et al., Ph.DThesis, University of Ottawa, 1992.) with 5 eq. of sodium azide in thepresence of tetrabutylammonium hydrogen sulfate (PTS) (1 eq.) in a 1:1mixture of CH₂Cl₂ and NaHCO₃ (saturated solution). After 3 hours, undervigorous stirring, the title compound was obtained as a pale yellowsolid in 97% yield. The characteristic ¹H-NMR [anomeric α-H; 4.56 ppm(³J_(H1′-H2′)=8.6 Hz)] and ¹³C-NMR [anomeric C-1′; 88.29 ppm] dataconfirmed the structure and was in agreement with published data. Thisreaction occurred with complete inversion (³J_(H1′-H2′)=8.6 Hz confirmedthe 1,2-trans-β-D-anomeric configuration) and therefore established thedesired stereochemistry.

EXAMPLE 4 Peracetylated-β-azidolactose

Following the procedures illustrated for Example 1 above, α-D-lactosylacid bromide was converted to the β-azidolactose. The title compound wasobtained as a pale orange oil in 92% yield. The characteristic ¹H-NMR[glucose anomeric α-H; 4.6 ppm (³J_(H1′-H2′)=8.8 Hz)] and ¹³C-NMR[glucose anomeric C-1′; 88.30 ppm] data confirmed the structure and wasin agreement with published data (Tropper et al., Synthesis, 1992, 7,618). The PTC reaction occurred with complete inversion as observed forthe peracetylated-β-azidogalactose.

EXAMPLE 5 Synthesis of peracetylated-β-aminogalactose

Hydrogenation of peracetylated-β-azidogalactose in the presence of alarge excess of Pd (10% on charcoal in wet methanol) at 40 p.s.i. for 1hour gave peracetylated-β-aminogalactoside as a pale yellow oil inquantitative yield. The characteristic ¹H-NMR data (the clean doublet ofthe anomeric proton in the azide shifted from 4.6 ppm to a multiplet at5.37-5.39 ppm in amine, indicating the absence of the anomeric azidegroup) and the mass spectrum analysis (CI-NH3: m/e 347 [M]⁺) confirmedthe structure.

EXAMPLE 6 Synthesis of peracetylated-β-aminolactose

As per the procedures illustrated in Example 5,peracetylated-β-azidolactose was hydrogenated under similar reactionconditions. The β-azide was hydrogenated in the presence of Pd (10% oncharcoal in wet methanol). After a simple celite filtration, the titlecompound was obtained in quantitative yield as a pale yellow oil. Thecharacteristic ¹³C-NMR data (the galactose anomeric carbon was at 101.14ppm and the glucose anomeric carbon shifted from 87.71 ppm in azide 4ato 84.48 ppm) and the mass spectrum analysis (FAB-NBA: m/e 636 [M+H]⁺)confirmed the structure.

The reduction reactions of Example 5 and 6 were adapted from Tropperibid. In this previously reported procedure stoichiometric amounts ofcatalyst to glycosyl azides was used. This procedure was found to bequite dangerous when synthesizing on large scales so the amount of Pdwas reduced to 0.2 to 0.4 eq. of Pd on carbon (10%). This amount wassufficient to reduce the glycosyl azides without affecting the acetates(no deacetylated galactosamines or lactosamines were detected by ¹H-NMRor ¹³C-NMR on the crude mixtures).

Due to the instability of both glycosylamines, no purification wasperformed prior to coupling with the linkers.

EXAMPLE 7 Attachment of N-Fmoc (9-fluorenylmethoxycarbonyl) Protectedε-aminocaproic Acid Linker to Peracetylated-β-aminogalactose

N-Fmoc-ε-aminocaproic acid (Scheme 8) is treated withO-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexa-fluorophosphate(HBTU, 1 eq.) in the presence of 1-hydroxybenzotriazole (HOBt) (1 eq.)and N-methylmorpholine (NMM) (1.5 eq.) in dry DMF. After 35 min. atr.t., peracetylated-β-aminogalactose was added and the reaction wasstirred for an additional 18 hours. The reaction mixture is concentratedand purified by chromatography to give the galactosylamide in a 45%yield. In this reaction, the anionic form of the acid attacks the HBTUforming the acyloxyphosphonium salt. This reactive salt is attacked byeither ionized HOBt or ionized acid, forming the benzotriazolyl activeester and symmetrical anhydride, respectively. The glycosylamine reactswith either of the reactive intermediates to yield the desired product.

The amide NH appeared as sharp doublet at 6.24 ppm (in CDCl₃) andcoupled strongly (from 2D-COSY) to the anomeric multiplet at 5.18-5.27ppm (3JNH-H1′=9.15 Hz). From ¹³C-NMR spectrum, the C-1′ anomeric carbonwas at 78.5 ppm. All the NMR data and the mass spectrum (FAB-NBA: m/e683 [M+H]⁺) confirmed the desired structure.

Other attempts to increase the coupling efficiency were investigated byusing the BOP reagent instead of HBTU. N-Fmoc-ε-aminocaproic acid wasreacted with BOP (1 eq.) reagent in the presence of HOBt (1 eq.) and NMM(1.5 eq.) in dry DMF. After 1 hour at r.t., the galactosylamine wasadded and 18 hours later the galactosylamide was obtained in a 51% yieldafter column chromatography.

The BOP method gave comparable yields and was the preferred reagentbeing approximately three times cheaper than HBTU. Triethylamine (TEA)was used instead of NMM since the latter is used in peptide couplingmethodology as a racemization suppressant. In the synthesis, there is noracemization and the use of triethylamine gave comparable results.

EXAMPLE 8 Attachment of N-Fmoc Protected Linker toPeracetylated-β-aminolactose

The attachment of an N-Fmoc protected linker toperacetylated-β-aminolactose is accomplished following the procedure ofExample 7. N-Fmoc-ε-aminocaproic acid was treated with BOP reagent (1eq.) in the presence of HOBt (1 eq.) and triethylamine (1.5 eq.) in dryDMF. After 1 hour at r.t., peracetylated-β-aminogalactose was added andthe reaction was stirred for an additional 24 hours. Following apreviously reported procedure (Coste et al., J. Org. Chem., 1994, 59,2437) a 10% citric acid solution was used instead of a saturatedsolution of NH₄Cl to efficiently extract the BOP and other by-productsfrom the organic layer of the BOP coupling reaction. The stronger acidicsolution extracted most of the BOP and the trace amounts of the activeester and the symmetric anhydride intermediates, which greatlysimplified the chromatographic purification. The lactosylamide productwas obtained after column chromatography in 51% yield. From the ¹H-NMRspectrum, the amide NH at 6.64 ppm appeared as a doublet(³J_(NH-H1′)=9.3 Hz). The glucose H1′ overlapped with the H3′ glucoseand a multiplet was observed between 5.11 and 5.21 ppm. The glucose H2′was a clean apparent triplet (J=9.8 Hz) which confirmed the β-amidelinkage through the 1,2-trans arrangement between H2′-H1′ and betweenH2′-H3′. The galactose and glucose anomeric carbons were at 100.8 and77.8 ppm, respectively. From FAB-MS, the pseudomolecular ion at 971([M+H]⁺) and some characteristic fragments (749 [M+H-Fmoc]⁺ and 619[M+H-{Fmoc-NH—CH₂(CH₂)₄—C(O)NH}]⁺) were in agreement with the assignedstructure.

EXAMPLE 9 Fmoc Cleavage ofPeracylated-β-N-[ε-(N-Fmoc)aminocaproic]-aminogalactose with Piperidineand TBAF

The peracylated-β-N-[ε-(N-Fmoc)aminocaproic]amino-galactose (productfrom Example 7) was treated with tetrabutylammonium fluoride (TBAF) (1.2eq., from a 0.1 M stock solution) (see Ueki et al., Tetrahedron Lett.,1997, 26, 560). After 2 hours at room temperature, the reaction mixturewas diluted with ethyl acetate and thoroughly washed with water. Afterchromatographic purification, peracylated-β-N-(ε-aminocaproic)aminogalactose was obtained in 29% yield. Characteristic ¹H-NMR data(sharp singlet at 2.11 ppm corresponding to the free amine NH due tofast exchange from hydrogen bonding and loss of all Fmoc proton signals)and mass spectrometry (FAB-NBA m/e: 461 [M+H]⁺, 100% relative intensity)confirmed the assigned structure.

EXAMPLE 10 Fmoc Cleavage ofPeracylated-β-N-[ε-(N-Fmoc)aminocaproic]-aminolactose with Piperidineand TBAF

The peracylated-β-N-[ε-(N-Fmoc)aminocaproic]amino-lactose (Example 8)was treated with TBAF (1.2 eq.) in dry DMF for 3 hours at roomtemperature. The free amine product was obtained after chromatography ina 55% yield. Characteristic ¹H-NMR data [a sharp singlet at 2.12 ppm,overlapping with the α-methylene protons to the amide linkage(lac-NHC(O)CH₂—), corresponded to the free amine NHD and the loss of allFmoc proton signals] and mass spectrometry (FAB-NBA m/e: 749 [M+H]⁺)confirmed the assigned structure.

EXAMPLE 11 Coupling of Glycosylamines with the N-carbobenzyloxy (Cbz)Protected Linkers

Commercially available N-Cbz-ε-aminocaproic acid was treated with BOPreagent (1 eq.) in the presence of HOBt (1 eq.) and triethylamine (1.6eq.) in dry DMF for 30 minutes. Peracetylated-β-aminogalactose (Example5) was added and stirring was maintained for an additional 48 hours. Theorganic layer was washed with a 10% citric acid solution to remove mostof the BOP and trace amounts of reactive intermediates. The coupledgalactosylamide having the Cbz protecting group on the ε-amino end ofthe amino caproic acid linker was obtained after chromatography in a 60%yield. From ¹H-NMR spectrum the amide NH appeared as a sharp doublet at6.43 ppm (³J_(NH-H1′)=9.3 Hz). The anomeric proton appeared as a doubletof doublets at 5.20 ppm (³J_(H1′-NH)=9.3 Hz, ³J_(H1′-H2′)=8.7 Hz) whichconfirmed the 1,2-trans arrangement of H1′-H2′ and the β N-glycosidicbond. The anomeric carbon was at 78.4 ppm in the ¹³C-NMR spectrum andthe mass spectra [Electrospray MS m/z: 617.4 [M+Na]⁺, 100; and highresolution (FAB-NBA) m/e: 595 [M+H]⁺, calc. for C₂₈H₃₉N₂O₁₂, 595.2424;found 595.2503] confirmed the assigned structure.

EXAMPLE 12 Coupling of Peracetylated-β-aminolactose withN-Cbz-ε-aminocaproic Acid

The ε-amino Cbz protected linked peracylated-β-aminolactose was preparedfollowing the procedures illustrated in Example 11. The product wasobtained in a 63% yield after column chromatography. From the ¹H-NMRspectrum the amide NH appeared as a sharp doublet at 6.14 ppm(³J_(NH-H1′(Glu))=9.3 Hz) and the anomeric glucose proton appeared at5.18 ppm as a doublet of doublets (³J_(H1′-H2′)=9.5 Hz, ³J_(H1′-NH)=9.3Hz) which confirmed the β-N-linkage. The galactose H1 appeared as abroad doublet at 4.43 ppm (³J_(H1′(gal)-H2′)=7.8 Hz). Mass spectrometry[Electrospray MS m/z: 905.5 [M+Na]⁺, 85.2 and 883.5 [M+H]⁺, 100; FAB-NBAm/e: 595 [M+H]⁺, calc. for C₄₀H₅₅N₂O₂₀, 883.3191; found 883.3348]confirmed the assigned structure.

EXAMPLE 13 Cleavage of the Cbz ofPeracylated-β-N-[ε-(N-Cbz)aminocaproic]aminogalactose

Peracylated-β-N-[ε-(N-Cbz)aminocaproic]amino-galactose was dissolved inethyl acetate/water/acetic acid solvent together with an equivalentweight of palladium (10% on activated charcoal as catalyst). The mixturewas agitated for 1 hour at a pressure of 40 p.s.i. hydrogen. The ¹H-NMRspectrum was very clean with a characteristic sharp singlet at 2.31 ppmcorresponding to the free amine. All the starting material was consumedand no Cbz proton signals were observed.

EXAMPLE 14 Cleavage of the Cbz ofperacylated-β-N-[ε-(N-Cbz)aminocaproic]aminolactose Hydrogenolysis

The cleavage of the Cbz group fromperacylated-β-N-[ε-(N-Cbz)aminocaproic]aminolactose was performedfollowing the procedures of Example 13. The ¹H-NMR spectrum was veryclean as observed in the galactosyl series of Example 13.

EXAMPLE 15 Synthesis of Tetrahydroxycholane, Cholane-type AnchoringBackbone

Cholic acid was reacted with borane (4 eq.) THF complex (BH₃.THF) in THFat 0° C. Recrystallization of the crude material from isopropanol gavethe tetrahydroxycholane in a 75% yield. The m.p. was found to be224-225° C. The methylene protons at position 24 (—CH₂OH) appeared as abroad triplet at 3.50 ppm in the ¹H-NMR spectrum. The carboxylate carbonof cholic acid, usually found at ^(˜)170 ppm, was absent in the ¹³C-NMRspectrum of the product. The NMR data and the mass spectrum(FAB-glycerol m/e 789 [2M+H]⁺ and 395 [M+H]⁺) confirmed the expectedstructure.

EXAMPLE 16 Synthesis of the t-butyldiphenylsilyl (TBDPS) Ether ofTetrahydroxycholane

Tetrahydroxycholane (Example 15) was reacted with TBDPSCl (1 eq.) in thepresence of imidazole (2 eq.) in dry DMF at 0° C. After purification bychromatography the TBDPS product was obtained in an 89% yield. From the¹H-NMR spectrum in CDCl₃, the methylene protons at position 24(—CH₂OSi—) appeared as a sharp triplet at 3.60 ppm (J=6.0 Hz). A sharpsinglet at 1.01 ppm corresponded to the tert-butyl group and the phenylprotons appeared between 7.33 and 7.68 ppm. The NMR data and the massspectrum (FAB-NBA m/e 655 [M+Na]⁺) confirmed the assigned structure.

EXAMPLE 17 Succinimide Activation of TBDPS Ether of Tetrahydroxycholane,Synthesis of the Active Ester

The TBDPS ether of tetrahydroxycholane (Example 16) was reacted withtriphosgene (2 eq.) in dry pyridine for 15 minutes with the temperaturemaintained at 0  C. N-hydroxysuccinimide (NHS) (10 eq.) was added andthe mixture became cloudy and thickened. Within 10 minutes of theaddition of the NHS the mixture started to become light pink and thecolor intensified slowly over the next 20-25 minutes to light red. After30 minutes the reaction mixture was poured into cold water and theactive ester precipitated out of the solution. Recrystallization from95% methanol gave a yield of 76% of the active ester. In the ¹H-NMRspectrum (FIG. 19), the 3-,7- and 12-α-CH protons appeared at 4.56-4.62(m), 4.87-4.88 (m) and 5.04 (s) ppm. The downfield shift of all the α-CHand the multiplet at 2.75-2.83 ppm, corresponding to the 12 succimidylmethylene protons [—(O)C—CH₂CH₂C(O)—] confirmed the tris activation. TheNMR data and the mass spectrum (electrospray MS m/z: 1078.6 [M+Na]⁺;1056.5 [M+H]⁺) confirmed the structure.

EXAMPLES 18-24 Preparation of Tris Peracylated Galactose and TrisPeracylated Lactose Conjugates of Formula

Example # R Q 18 peracylated galactose TBDPS 19 peracylated lactoseTBDPS 20 peracylated galactose OH 21 peracylated lactose OH 22-24peracylated lactose DMT 25 peracylated lactose OH

EXAMPLE 18 Tris Coupling ofPeracylated-β-N-(ε-aminocaproic)amino-peracylated Galactose to the NHSActive Ester

Active ester (500 mg) was reacted withperacylated-β-N-(ε-aminocaproic)aminoperacylated galactose in dry DMF.After 15 hours at r.t., the reaction mixture was poured into ice coldwater and the resulting precipitate was collected and dried underreduced pressure for 24 hours. The 960 mg of product, collected fromrunning the above reaction sequence twice and combining the products,was dissolved in dry pyridine containing acetic anhydride (20 eq.). Themixture was allowed to stir for twelve hours and then was poured into200 mL of ice water. The resulting pale yellow precipitate was isolatedand dried to give a ^(˜)90% yield. Further purification was not neededas only one spot was obtained by TLC with an R_(f)=0.4 in 5%MeOH/CH₂Cl₂. The ¹H-NMR spectrum was quite complex. The compound wasvery soluble in CDCl₃ and CD₂Cl₂, but strangely yielded broad andundefined proton resonances. We do not understand the factors involved.One can speculate that such molecules fold, interact and relaxdifferently from non-polar to polar solvents. Only DMSO-d₆ could be usedto obtain high resolution spectrum. The three anomeric protons appearedat 5.31 ppm as an apparent triplet. Although a J value can be extracted(J=9.3 Hz), it is misleading to use this coupling constant since threetriplets are superimposed. At 2.85 to 3.01 ppm, a complex set ofmultiplets integrated for 6H and corresponded to the 3 [—CH₂—NH—C(O)O—]protons. In the acetyl CH₃ region between 1.92 and 2.07 ppm, 5 singletsand one multiplet integrated for 36H and correlated well withtrisubstitution. Both the NMR data and the electrospray mass spectra{(iPrOH/DMF/TFA) m/z: 2092.4 [M+H]⁺, 1588.1 [M+H-C₂₁H₃₁N₂O₁₂]⁺, 1083.8[M+H-C₄₂H₆₂N₄O₂₄]⁺ and 461 [M+H-C₈₃H₁₁₉N₄O₂₇Si]⁺; (DMF/TFA/AcOH) m/z:2092.4 [M+H]⁺; (DMF/CsI) m/z: 2224.2 [M+Cs]²⁺} confirmed the assignedstructure.

EXAMPLE 19 Tris Coupling ofPeracylated-β-N-(ε-aminocaproic)amino-peracylated Lactose to the NHSActive Ester, Tris-Coupled Peracylated Galactose Conjugate

The NHS active ester was tris coupled toperacylated-β-N-(ε-aminocaproic)aminoperacylated galactose (Example 14)following the procedures illustrated in Example 18. NHS active ester wasreacted with lactosylamine (3.3 eq.) in dry DMF at room temperature.After 18 hours, the reaction mixture was poured into ice water and thetrisubstituted lactosyl glycoconjugate was obtained in 87% yield. Themono-deacetylated material (less than 10% by electrospray mass spectra)was reacetylated by treatment with acetic anhydride.

The ¹H-NMR spectrum of the title compound was complex. Three sets ofcomplex multiplets at 5.10-5.28 ppm integrating for 12H corresponded tothree glucose anomeric protons, three H4′ (peracylated galactose), threeH3′ (glucose) and three H3′ (peracylated galactose), as deduced from2D-COSY spectra. The three H1′ (glucose) overlapped and appeared as oneapparent triplet with ³J-coupling values (9.0 and 12.0 Hz) which are notaccurate for any of the three anomers. Between 0.68 and 2.08 ppm, acomplex area was found to integrate to ^(˜)170H. The integrationaccounted for all the stern H and —CH₃ groups, 9 —CH₂— caproic groups, 3—CH₂— groups adjacent to the amide linkage, and 63H for the 21 acetyl—CH₃ groups. From ¹³C-NMR, the peracylated galactose anomeric carbon wasat 99.78 ppm and the glucose anomeric carbon at 76.54 ppm. The NMR dataand the mass spectrum [electrospray (DMF) m/z: 2978.2 [M+Na]⁺; 2186.2[M+Na—C₃₃H₄₇N₂O₂₀]^(•+)] confirmed the assigned structure.

EXAMPLE 20 Fluoride-promoted Desilylation of Tris-Coupled PeracylatedGalactose Conjugate

The peracylated galactose conjugate (title compound of Example 18) wasdesilylated by treatment with TBAF (25 eq.) in the presence of AcOH (12eq.) in dry THF. After 1 hour at room temperature additional TBAF (13eq.) was added and the progress of the reaction was closely monitored.After 30 minutes from the second addition of TBAF, there was stillstarting material left and a third spot (below the product) appeared.The reaction mixture was immediately poured into ice water to avoidfurther deacetylation of the product. A sticky precipitate developed andwas extracted in ethyl acetate.

Several water washes were performed to remove the excess TBAF and aftersilica gel column chromatography using 2-2.5% MeOH in CH₂Cl₂, threefractions were obtained. The first consisted of some starting materialwhich overlapped with the desilylated product (two spots; topR_(f)=0.28, bottom R_(f)=0.21 in 5% MeOH/CH₂Cl₂; 19%). The middlefractions consisted essentially of desilylated tris-coupled peracylatedgalactose conjugate (one middle spot; R_(f)=0.21 in 5% MeOH/CH₂Cl₂; 55%)and the last fraction was a mixture of desilylated product anddeacetylated by-products (^(˜)15%). The TBAF method was sufficientlyacceptable for the synthesis of 500 mg of pure desilylated tris-coupledperacylated galactose conjugate, required for biological testing.

The ¹H-NMR in DMSO-d₆ (middle spot, 23) was very sharp and severalassignments were made (FIG. 22). Three carbamate NH's appeared as broadmultiplets between 6.60 and 6.84 ppm. The three H1′ protons appeared at5.32 ppm as a sharp apparent triplet. The H2′ protons appeared between4.97 and 5.02 ppm as a multiplet with triplet-like character.

The 500 MHZ ¹H-NMR and 2D-COSY spectrum of the desilylated tris-coupledperacylated galactose conjugate showed 24-OH groups appeared between3.30 and 3.40 ppm as a very intense broad singlet which overlapped withresidual water. The t-butyl and phenyl group H's were absent. The aboveNMR data and the electrospray mass analysis [(DMF/AcOH) m/z: 1876.7([M+H+Na]⁺, 100), 1854.4 ([M+H]⁺, 5.15), 1371.8([M+H-1-C₂₁H₃₁N₂O₁₂+Na]^(•+), 9.82), 1349.8 ([M+H-1-C₂₁H₃₁N₂O₁₂]^(•+),9.82), 527.2 ([M+H-C₆₆H₁₀₁N₄O₂₅+Na]^(•+), 5.73), 503.4([M+H-C₆₆H₁₀₁N₄O₂₅]^(•+), 2.54)] confirmed the structure assigned forthe desilylated tris-coupled peracylated galactose conjugate.

EXAMPLE 21 Fluoride-promoted Desilylation of Tris-Coupled PeracylatedLactose Conjugate

The tris-conjugated peracylated lactose conjugate (Example 19) wasdesilylated using the procedures of Example 20. The desilylatedtris-coupled peracylated lactose conjugate was obtained in 37% yield.Some product overlapped with 20% of starting material and withappreciably more deacetylated by-products (^(˜)25%). The ¹H-NMR of theproduct (middle spot) in DMSO-d₆ was very sharp and several protons wereassigned in conjunction with the 2D-COSY spectrum.

The 500 MHZ ¹H-NMR and 2D-COSY spectrum showed three carbamate NH's thatappeared as broad multiplets between 6.59 and 6.84 ppm. Between 5.09 and5.33 ppm, three complex multiplets integrated for 12 protons. Afterevaluation of the 2D-COSY spectra for this area the signals wereassigned to three sets of H1′ [glucose], H4′ [peracylated galactose],H3′ [glucose] and H3′ [peracylated galactose]. The H2′ peracylatedgalactose proton appeared as an apparent doublet of doublets at 4.83ppm. The 24-OH group appeared between 3.28 and 3.33 ppm as a veryintense broad singlet which overlapped with residual water. The t-butyland phenyl group H's were absent. From ¹³C-NMR and 2D-HeteronuclearMultiple Quantum Coherent (HMQC) spectra, many carbons were assignedwithin the carbohydrate and linker regions. The three anomeric glucosecarbons were at 76.50, 76.52 and 76.55 ppm. The peracylated lactoseanomeric carbons were at 99.80, 99.77 and 99.70 ppm. The 24-CH₂OH carbonwas at 61.17 ppm. The above NMR data and the electrospray mass analysis[(DMF/AcOH) m/z: 2740.1 ([M+Na]⁺, 100.0), 2756.1 ([M+K]⁺, 11.8)]confirmed the structure assigned for the desilylated tris-coupledperacylated lactose conjugate.

EXAMPLE 22 Dimethoxytrityl Tetraol

The desilylation of the tris-coupled peracylated lactose conjugate usingthe above TBAF method was more problematic than for the galactosylconjugate probably because of the higher bulkiness and the possibleincreased hindrance of the TBDPS group rendering it inaccessible tofluoride ion attack at low TBAF concentrations. To eliminate therestrictions associated with this route another method was used tosynthesize the tris-coupled peracylated lactose conjugate.

Tetraol (prepared in Example 15) was reacted with 1.1 eq. ofdimethoxytrityl chloride (DMT-Cl) in the presence of 0.1 eq. of DMAP indry pyridine at 0° C. After 10 minutes the reaction mixture was allowedto warm to room temperature. The reaction was allowed to proceed for 6hours and the crude material obtained after workup was purified bysilica gel column chromatography to give the title compound in a 64%yield.

The methylene protons at position 24 (—CH₂O—DMT) appeared as a multipletbetween 2.86 and 2.94 ppm in the ¹H-NMR spectrum. The 7- and12-equatorial —CH(OH)— appeared as singlets at 3.59 and 3.76 ppm,respectively. The 3-axial —CH(OH)— appeared as a multiplet between 3.14and 3.19 ppm. The two methoxy CH₃ appeared as a sharp singlet at 3.71ppm and the phenyl protons appeared between 6.8 and 7.3 ppm. The NMRdata and the mass spectrum {FAB-NBA m/e 697 [M+H]⁺; 303[M+H-(C₂₄H₄₁O₄)]⁺, 100} confirmed the assigned structure.

EXAMPLE 23 Synthesis of DMT-Protected Tris-NHS Cholane Derivative,DMT-Protected Active Ester

Dimethoxytrityl tetraol was reacted with triphosgene (2 eq.) in drypyridine at 0° C. After 15 minutes at 0° C., N-hydroxysuccinimide (NHS,10 eq.) was added. The reaction mixture became cloudy and thickened.Within 10 minutes the mixture started to become light red and at 15minutes the reaction mixture was poured into cold water. The esterprecipitated out of the solution and after drying, the active ester wasobtained in 89% yield.

The 3-,7- and 12-α-CH protons appeared at 4.55-4.61 (multiplet), 4.87(singlet) and 5.03 (singlet) ppm in the ¹H-NMR spectrum. The downfieldshift of all the α-CH and the multiplet at 2.78-2.83 ppm, correspondingto the 12 succimidyl methylene protons [—(O)C—CH₂CH₂C(O)—] confirmed thetris activation. The NMR data and the mass spectrum [(FAB-NBA) m/e:1143.38 ([M+Na]⁺, 0.4), 1120.47 ([M+H]⁺, 1.1), 303.21([M+H-C₃₉H₅₀N₃O₄]⁺, 100)] confirmed the assigned structure.

EXAMPLE 24 Preparation of DMT-Protected Lactosylated Conjugate

The DMT-active ester was reacted with lactosylamine (3.3 eq.) in dry DMFat room temperature. After 120 hours, the reaction mixture was pouredinto ice water and the a 1:1 mixture of tritylated and detritylatedtris-coupled peracylated lactose conjugate was obtained in >80% yield.The observed detritylation was not problematic since the following stepwas the trifluoroacetic acid treatment (detritylation). A small amountof the mixture was subjected to column chromatography and the two spotswere separated and individually characterized by NMR and electrospraymass spectrometry. The remainder of the mixture was completelydetritylated in the following step.

EXAMPLE 25 Cleavage of the DMT-Group with Trichloroacetic Acid (TCA)

The mixture of protected and deprotected tris substituted peracylatedlactose conjugates (Example 23) was reacted with 6% TCA in dry CH₂Cl₂ atroom temperature. The reaction mixture was quenched with methanol andtriethylamine after 1.5 hours. Flash column chromatography using aincreasingly polar eluent mixture (CH₂Cl₂ to 2.5% MeOH/CH₂Cl₂ to 5%MeOH/CH₂Cl₂) gave the deblocked tris peracylated lactose conjugate in80% yield. Electrospray MS and NMR were identical to those reported forthe tris peracylated lactose conjugate previously obtained in Example 21using the TBAF method. Using the DMT protection scheme, 500 mg of puretris peracylated lactose conjugate was prepared as required forbiological testing.

EXAMPLE 26 General Method of Incorporating Tris-Coupled PeracylatedGalactose Conjugate (Example 20) Into an Oligonucleotide or anOligonucleoside

The tris-coupled peracylated galactose conjugate of Example 20 isactivated with disuccinimidyl carbonate (DSC, Fluka) to give thecarbamate derivative. The carbamate is then treated with4-amino-2-hydroxy butanol (prepared as per J. Am. Chem. Soc. 109, 3089,1987) to give the corresponding carbamate. The primary alcohol functionis then treated with dimethoxytrityl chloride/pyridine to give the DMTderivative.

The DMT derivative is then phosphitylated using2-cyanoethyl-N,N-diisopropylchlorophosphoramidite in the presence ofdiisopropyl ethyl amine in methylene chloride as the solvent. Theresulting DMT phosphoramidite derivatized tris-coupled peracylatedgalactose conjugate is incorporated at the terminal 5′-OH or 3′-OH of anoligonucleotide or an oligonucleoside.

Alternatively the DMT phosphoramidite is coupled to a free 5′-OHposition of a nucleotide, nucleoside, oligonucleotide or anoligonucleoside. Followed by deblocking of the DMT group the freeprimary hydroxyl is available for coupling to a further nucleotide,nucleoside, oligonucleotide or an oligonucleoside to effectincorporation of the conjugate at an internal position.

EXAMPLE 27 Incorporation of Tris-Coupled Peracylated Lactose Conjugate(Example 21) Into an Oligonucleotide or an Oligonucleoside

Both the DMT and DMT phosphoramidite tris-coupled peracylated lactoseconjugate (Example 21) are prepared as per the procedure illustrated inExample 26. The DMT phosphoramidite is further incorporated onto the 3′or 5′ position or at an internal position of an oligonucleotide or anoligonucleoside.

EXAMPLE 28 General Method, Attachment of the DMT-Tris-CoupledPeracylated Galactose Conjugate (Example 26) Onto Solid Support

DMT-tris-coupled peracylated lactose conjugate (Example 26) issuccinylated using succinic anhydride (1.5 equivalents), triethylamine(1 equivalent), 4-dimethylamino pyrimidine (0.5 equivalent) in anhydrous1,2-dichloroethane at 50° C. (as per the procedure of Kumar et al.,Nucleosides and Nucleotides, 1993, 12, 565-584). After workup theresulting succinate is dried under vacuum at room temperature. Thesuccinate is then condensed with controlled pore glass that has beenpre-acid washed (CPG Inc., New Jersey) (as per the procedure of Bayer etal., Z. Naturforsch, 1995, 50b, 1096-1100). The amino-functionalized CPGis suspended in anhydrous DMF and treated with 1 equivalent of thesuccinate, 1 equivalent of TBTU (2-(1H-benzotriazole-1-yl)-1,1,3,3-tetra-methyluronium tetra-fluoroborate)and 2 equivalents of N-methyl morpholine (NMM). After 12 hours ofshaking the functionalized CPG, support is filtered and washed with DMF,methylene chloride and methanol. The final rinse is carried out withether and the derivatized solid support is obtained after drying undervacuum.

EXAMPLE 29 Attachment of the DMT-Tris-Coupled Peracylated LactoseConjugate (Example 27) Onto Solid Support

The DMT protected tris-coupled peracylated lactose conjugate (Example27) is attached to a solid support as per the procedures illustrated inExample 28.

EXAMPLE 30 Preparation of Tetra-Coupled Cyclohexanone PeracylatedCarbohydrate Conjugate

Cyclohexanone is treated with base (KOH) followed by four equivalents ofacrylonitrile. The resultant tetracyano compound is subjected to aWitting reaction (—C═O to —C—CH₂) followed by hydroboration to convertthe olefin to a primary alcohol. Catalytic hydrogenation with Pd/H₂ toconvert the cyano groups to methylamino groups will give thetetra-aminopropyl-cyclohexylhydroxymethyl compound having 4 linkergroups attaching 4 amino groups. Thetetra-aminopropyl-cyclohexylhydroxymethyl compound is then condensedwith carbohydrate groups using either a reductive amination route (e.g.,acylated lactose) or isocyanate condensation to give thetetra-carbohydrate substituted-aminopropyl-cyclohexylhydroxymethylcompound. The free hydroxyl group is extended with a linker such as4-amino-2-hydroxy-butanol in a similar manner as illustrated above inExample 26. Introduction of the primary and secondary hydroxyl groupsenables the preparation of the DMT or DMT phosphoramidite conjugates.The conjugates can be incorporated internally or at the 3′ or 5′terminus of a nucleotide, nucleoside, oligonucleotide or anoligonucleoside as illustrated in Example 26 above.

EXAMPLE 31 Preparation of Tetra-Coupled-5-Aminoisophthalic AcidConjugate

The above compound prepared as per the procedure of Hayashi et al., J.Am. Chem. Soc., 1998, 120, 4910-4915) is treated with HBr/HOAC andcondensed with pentafluoro-phenyl ester of TBDPS-O—(CH₂) ₆—COOH(Pg=TBDPS). The resulting tetraester is refluxed with KOH/THF/MeOH togive tetracarboxylic acid compound. Either of the carbohydratederivatives prepared in Examples 9 and 10 are condensed with thetetracarboxylic acid compound above using TBTU/NMM to give thetetra-sugar conjugated compound. The silyl protecting group is removedby treatment with tBuN⁺F⁻ and the alcohol is phosphitylated to give theconjugate phosphoramidite. The conjugate phosphoramidite is used toconjugate the carbohydrate cluster to an oligonucleotide or anoligonucleoside as illustrated in Example 26 above.

Alternatively, the free hydroxyl obtained after removal of the TBDPSgroup can be activated using an activating agent followed by treatmentwith a linker group having one functionality to couple with theconjugate group and also having a primary and a secondary hydroxylgroup. Introduction of the primary and secondary hydroxyl groups enablesthe preparation of the DMT or DMT phosphoramidite conjugates. Theconjugates can be incorporated internally or at the 3′ or 5′ terminus ofa nucleotide, nucleoside, oligonucleotide or an oligonucleoside asillustrated in Example 26 above.

What is claimed is:
 1. A complex having the structure:

wherein: Q_(a) is a nucleoside, nucleotide, an oligonucleoside, anoligonucleotide or a pro-drug form of said nucleoside, nucleotide,oligonucleoside or oligonucleotide, or a peptide, protein, a moleculethat can bind RNA, an antibiotic or an antibacterial compound; and eachR_(a), R_(b), and R_(c) independently, is a cell surface receptorligand.
 2. The complex of claim 1 wherein Q_(a) is a nucleoside,nucleotide, oligonucleoside or an oligonucleotide.
 3. The complex ofclaim 2 wherein Q_(a) is an oligonucleotide.
 4. The complex of claim 3wherein said oligonucleotide is a modified oligonucleotide.
 5. Thecomplex of claim 4 wherein said modified oligonucleotide comprises oneor more modified pyrimidine or purine base, substitution at a 2′position, alteration of an inter-nucleoside linkage, carbohydrate ringsubstitutions or a positional variation.
 6. The complex of claim 1wherein each of said cell surface receptor ligands is the same.
 7. Thecomplex of claim 1 wherein each of said cell surface receptor ligands isa carbohydrate.
 8. The complex of claim 7 wherein each of saidcarbohydrates is a monosaccharide or a polysaccharide.
 9. The complex ofclaim 8 wherein each of said monosaccharide or polysaccharide isselected from the group consisting of galactose, lactose,N-acetylgalactosamine, mannose and mannose 6-phosphate.
 10. The complexof claim 9 wherein each of said monosaccharide or polysaccharide isgalactose or lactose.